The present invention relates to peptides which mimic certain of the biological activities of Neuropeptide Tyrosine (NPY) at specific NPY receptors that modulate neuronal release of physiologically active substances. These receptors are often located on neurones at neuroeffector junctions and, in some tissues and species, have been classified as of the NPY Y2 receptor subtype. In addition, the present invention relates to pharmaceutical compositions including, as the active ingredient, these peptides and to methods of treatment involving the administration of these compositions.
Neuropeptide Y, a 36 amino acid peptide belonging to the pancreatic polypeptide family, was first isolated from porcine brain in 1982 (Tatemoto et al., 1982) and has since been identified in most sympathetic postganglionic neurons innervating the cardiovascular system, where it is co-localised with noradrenaline (Potter, 1988). In the cardiovascular system it raises blood pressure by an action on postjunctional neuropeptide Y receptors (Dahl{overscore (o)}ff et al., 1985; Potter, 1985; Revington et al., 1987; Potter and McCloskey, 1992) and inhibits neurotransmitter releasexe2x80x94both acetylcholine (Revington et al., 1987; Warner and Levy, 1989) and noradrenaline (Edvinsson, 1988)xe2x80x94by acting on prejunctional neuropeptide Y receptors. Receptors for neuropeptide Y are also located on sensory nerve terminals and their activation can modulate local neurogenic responses (Grundemar et al., 1990;1993). These two receptor subtypes have been called neuropeptide Y Y1(postjunctional) and neuropeptide Y Y2 (prejunctional) on the basis of the different responses to a truncated analog of the related peptide YY-(13-36), when compared with neuropeptide Y in in vitro assay systems (Wahlestedt et al., 1986). Apart from these historically well-defined neuropeptide Y receptors the existence of a number of other subtypes (Y3, Y4, Y5, Y6 and Y7) have been suggested on pharmacological grounds and details of the cloning of receptors corresponding to Y1, Y2, Y4 and Y5 have been published (Herzog et al., 1992; Gerald et al., 1995; Bard et al., 1995; Gerald et al., 1996). The distribution and physiological significance of these various receptor subtypes has yet to be defined. Although some controversy has existed about the selectivity of truncated forms of neuropeptide Y for one or other receptor subtype (Potter et al., 1989), the emerging picture supports the initial classification into pre- and postjunctional receptor subtypes. Cell lines have been developed which express one or other neuropeptide Y receptor subtype and the development of receptor-selective analogs of neuropeptide Y has focussed mainly on binding characteristics in these cell lines (Sheikh et al., 1989; Aakerlund et al., 1990; Fuhlendorff et al., 1990). More recently, a cDNA encoding the neuropeptide Y Y1 receptor has been cloned and cell lines expressing the cloned receptor have been analysed for both specific binding of neuropeptide Y analogs (Herzog et al., 1992) and functional responses elicited by specific analogs. From such binding studies, combined with subsequent studies in vivo, two analogs have been classified as acting specifically on the postjunctional (neuropeptide Y Y1) receptor. These neuropeptide Y Y1 selective analogs, (Pro34) neuropeptide Y and (Leu31, Pro34) neuropeptide Y, mimic the action of neuropeptide Y in raising blood pressure, and also share similar binding to cell lines expressing only neuropeptide Y Y1 receptors e.g. the human neuroblastoma cell line SK-N-MC and fibroblast lines expressing the cloned neuropeptide Y Y1 receptor (Herzog et al., 1992). Neither exhibits the neuropeptide Y Y2 receptor action of inhibiting cardiac vagal action in vivo, a manifestation of inhibition of acetylcholine release (Potter et al., 1991; Potter and McCloskey, 1992).
Activation of neuronal prejunctional NPY receptors generally inhibits nerve activity, reducing the release of neurotransmitters in response to nerve impulses and in response to local factors acting to release neurotransmitters (Wahlestedt et al., 1986).
NPY-containing neurons are evident in the nasal mucosa of various species including man, often associated with glandular acini and blood vessels (Baraniuk et. Al., 1990; Grunditz et. al., 1994). Stimulation of the parasympathetic nerve supply to the nasal mucosa (vidian nerve) in dogs increases blood flow in the region and the major part of this effect is atropine resistant. Intravenous administration of NPY reduces vasodilitation due to parasympathetic nerve stimulation, an effect that was not mimicked by the NPY Y1-selective agonist [Leu31, Pro34]NPY, but was mimicked by administration of the NPY Y2-receptor agonist N-acetyl[Leu28,Leu31]NPY(24-36) (Lacroix et al., 1994). This is consistent with a prejunctional NPY Y2-like receptor-mediated inhibition of transmitter release from parasympathetic nerve terminals.
The prejunctional or neuropeptide Y Y2 receptor classification was based on actions of peptide YY (13-36) but in many systems this molecule, as well as neuropeptide Y-(13-36), does exhibit pressor activity (Rioux et al., 1986; Lundberg, et al., 1988; Potter et al., 1989). This has been interpreted by some to indicate that in some vascular beds there are two types of neuropeptide Y receptor (both neuropeptide Y Y1 and neuropeptide Y Y2) on postjunctional membranes (Schwartz et al., 1989). However the lack of selectivity of these molecules may be due to retention of partial agonist activity on Y1 receptors, which permits them to evoke a reduced functional response. We have previously described a 13-36 analog of neuropeptide Y, (Leu17, Glu19, Ala21, Ala22, Glu23, Leu28, Leu31) neuropeptide Y-(13-36) (ANA neuropeptide Y-(13-36)) which displayed prejunctional activity equivalent to the whole neuropeptide Y molecule in studies in vivo (Potter et al., 1989). However, this analog still retained significant pressor activity, or neuropeptide Y Y1 receptor-mediated interactions.
We have also previously described analogs of neuropeptide Y which mimic the action of neuropeptide Y in inhibiting cardiac vagal action but have no pressor action. Consistent with these functional responses are binding studies with one analog, N-acetyl [Leu28, Leu3] neuropeptide Y-(24-36), which showed significant affinity for the neuropeptide Y Y2 receptor subtype expressed on the human neuroblastoma cell line SMS-KAN, but no affinity for the neuropeptide Y Y1 receptor type expressed on the human cell line SK-N-MC (Potter et al., 1994). In addition, this analog did not stimulate the human neuropeptide Y Y1 receptor expressed in fibroblast cells to induce an increase in cytosolic calcium, although the receptor responds to intact neuropeptide Y.
The present inventors have now developed novel peptides that mimic responses attributed to activation of neuropeptide Y Y2-like receptors. In in vitro assays these agonists show high affinity at neuropeptide Y Y2 receptors and have low affinity for NPY Y1 receptors. In in vivo assays the new agonists exhibit similar or enhanced NPY Y2-receptor-like agonist activity when compared with N-acetyl [Leu28, Leu31] neuropeptide Y-(24-36) and they show no pressor or Y1-receptor activiy at doses eliciting maximal neuropeptide Y Y2-like agonist action.
Accordingly, in the first aspect the present invention consists in a ligand for a neuropeptide Y receptor having the formula:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15
wherein
X1 is H, R1-CO or one or two naturally occurring amino acids
X2 is Leu, Ile, Val, Nie, Sar, Gly, Ala, Aib, D-Leu, D-Ile, D-Val, D-Ala or D-Nle;
X3 is Arg, Lys, Orn, Ala, Dbu or His;
X4 is His, Lys, Arg, Ala, Gly, Ser, Thr, Asn, Gln or Aib;
X5 is Tyr, Phe, Ala, Gly, Ser, Thr, Asn, Gln or Aib;
X6 is Leu, Ile, Val, Ala, Arg or Nle;
X7 is Asn, Ala or Gln;
X8 is Leu, Ile, Val, Ala, Aib or Nle;
X9 is Leu, Ile, Val, Ala, Aib or Nle;
X10 is Thr, Ala or Ser;
X11 is Arg, Lys or Orn;
X12 is Gln, Pro or Asn;
X13 is Arg, Lys or Orn;
X14 is Tyr, Phe, His, Trp, D-Tyr, D-Phe, D-His or D-Trp;
X15 is OH, NH2, NHR2, NR3R4 or one or two naturally occurring amino acids with the terminal amino acid being in the normal or amide form;
wherein R1, R2, R3 and R4 are independently alkyl groups, straight, branched or alicyclic in structure; and
wherein at least one of X2 to X10 is Ala.
Abbreviations:
Sar: Sarcosine or N-methylglycine
Aib: Aminoisobutyric acid
Orn: Ornithine
Dbu: Diaminobutyric acid
In a preferred embodiment, at least two groups selected from X2, X4, X5, X8 and X9 are Ala. In a particularly preferred embodiment, X2 and X5 are Ala.
In a further preferred embodiment, R1 is an alkyl group selected from methyl, ethyl, n-butyl, t-butyl, cyclohexyl and other alkyl groups with 10 or less carbon atoms.
In a further preferred embodiment, R2, R3 and R4 are alkyl groups selected independently from methyl, ethyl, isopropyl, n-butyl, cyclohexyl and other alkyl groups with 10 or less carbon atoms.
In a preferred embodiment of this invention the neuropeptide Y receptor is a neuropeptide Y Y2-like receptor. By xe2x80x9cneuropeptide Y Y2-like receptorxe2x80x9d we mean a receptor which shares pharmacological properties with the human neuropeptide Y Y2 receptor. Such receptors may modulate the release of neurotransmitters such as acetylcholine and noradrenaline and may modulate the release of effectors from sensory nerves. Some NPY receptor subtypes, for example Y5, can be activated by ligands with high potency at NPY Y2 receptors but low potency at NPY Y1 receptors (Gerald et al., 1996) and are therefore Y2-like. In a most preferred embodiment, the receptor is a neuropeptide Y Y2 receptor.
The ligands of the invention may be in multimeric form; ie. they may be in dimeric or trimeric form.
It will be appreciated by those skilled in the art that a number of modifications may also be made to the peptides of the present invention without deleteriously affecting the biological activity of the peptide. This may be achieved by various changes, such as insertions and substitutions, either conservative or non-conservative in the peptide sequence where such changes do not substantially decrease the biological activity of the peptide.
Modifications of the peptides contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5xe2x80x2-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-bitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid; 2-thienyl alanine and/or D-isomers of amino acids.
As a further example, it is possible in the present invention to replace the residue at X14 by other non-natural amino acids with hydrophobic side chains such as Cha (beta-cyclohexyl-L-alanine), Nal (beta-(2-naphthyl)-alanine), Phg (L-phenylglycine), Tic (L-1,2,3,4-tetrahydroisoquinoline 3-carboxylic acid), Thi (beta-(2-thienyl)-L-alanine), or their D-isomers, without substantially altering the biological activity.
It may also be possible to add various groups to the peptide of the present invention to confer advantages such as increased potency or extended half life in vivo without substantially decreasing the biological activity of the peptide. It is intended that such modifications to the peptide of the present invention which do not result in a decrease in biological activity are within the scope of the present invention.
The ligands of the present invention may be useful in treatment of the following conditions:
Conditions related to inflammation (such as nasal inflammation, rhinitis including allergic and vasomotor, asthma, arthritis).
Conditions related to neurogenic inflammation (such as migraine, headache, inflammation of the eye, rhinitis, etc.).
Rhinitisxe2x80x94vasomotor rhinitis.
Respiratory diseases (such as pulomonary congestion, asthma, upper respiratory tract inflammation).
Sleep disorders.
Conditions related to increased sympathetic nerve activity.
Disorders related to sexual dysfunction and reproductive disorders.
Disorders or diseases pertaining to the heart, blood vessels or the renal system (such as vasospasm, heart failure, shock, cardiac hypertrophy, increased blood pressure, angina, myocardial infarction, sudden cardiac death, arrhythmia, peripheral vascular disease, renal failure, etc.).
Conditions related to increased sympathetic nerve activity (e.g. during or after coronary artery surgery, and operations and surgery in the gastrointestinal tract).
Cerebral diseases and diseases related to pain or nociception.
Diseases related to the central nervous system (e.g. cerebral infarction, neurodegeneration, epilepsy, stroke, cerebral vasospasm, depression, anxiety or dementia).
Diseases related to abnormal gastrointestinal motility and secretion (e.g. Crohn""s disease).
Diseases and conditions affecting the urinogenital system (e.g. urinary incontinence).
Abnormal drink and food intake disorders (such as obesity, anorexia, bulimia etc.).
In a second aspect, therefore, the present invention consists in a composition for use in relieving nasal congestion or treating vasoconstriction predisposing to acute renal failure, anti-hypertensive conditions, cardiovascular disorders, conditions related to pulmonary congestion, inflammation, neurogenic inflammation, sleep disorders, conditions related to increased sympathetic nerve activity, diseases related to the central nervous system, conditions related to pain or nociception, diseases related to gastrointestinal motility and secretion, obesity, or Alzheimer""s disease, or as an anti-psychotic, the composition including the peptide of the first aspect of the present invention and a pharmaceutical carrier.
In a third aspect the present invention consists in a method of relieving nasal congestion, attenuating cardiac vagal action, treating vasoconstriction predisposing to acute renal failure, hypertension, cardiovascular disorders, conditions related to pulmonary congestion, inflammation, neurogenic inflamation, sleep disorders, conditions related to increased sympathetic nerve activity, diseases related to the central nervous system, conditions related to pain or nociception, diseases related to gastrointestinal motility and secretion, obesity, or Alzheimer""s disease in a subject comprising administering to the subject an effective amount of the composition of the second aspect of the present invention.
In a preferred embodiment of the third aspect of the present invention the subject is suffering from nasal congestion, pulmonary congestion or vasoconstriction predisposing to acute renal failure. In a further preferred embodiment the composition is administered as a nasal spray.
The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). It may also be inhaled (dry or in solution) into the lungs. Depending on the route of administration, the active ingredients may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, low lipophilicity of peptides might allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer peptides by other than parenteral administration, they could be coated by, or administered with, a material to prevent its inactivation. For example, peptides may be administered in a solvent, in liposomes, or co-administered with enzyme inhibitors. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposomes include water-in-oil emulsions as well as conventional liposomes.
The active compounds may also be administered parenterally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients. In the case of sterile powders for use as such or for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.